The CFTR Cl^– channel activates anion exchangers of the SLC26 family through direct intermolecular interactions (Ko et al. 2004). This may account for the defective HCO₃^– secretion observed in epithelia affected by cystic fibrosis. The Calu-3 cell line, derived from human airway submucosal glands, is a good model for investigating CFTR-dependent HCO₃^– secretion. Our aim was to characterize the apical membrane anion exchanger in these cells and examine the effects of elevated intracellular cAMP on its activity. Calu-3 cells were grown to confluence on Transwell polyester filters (Corning) and loaded with the pH-sensitive fluoroprobe BCECF. Filters were superfused with HCO₃^–-buffered solutions ((mM) 144 Na⁺, 5 K⁺, 1 Ca²⁺, 1 Mg²⁺, 123 Cl^–, 25 HCO₃^–, 1 SO₄^2-, 5 Hepes, 10 glucose) and maintained at 37°C on the stage of an inverted fluorescence microscope. Intracellular pH (pH_i) was recorded by standard microfluorometric techniques. Substitution of Cl^– by gluconate on the basal side of the Calu-3 cells led to an increase in pH_i of 0.28 ± 0.04 (mean ± s.e.m., n = 4, P < 0.01 by paired t test). This was abolished by pre-treatment with 0.1 mM DIDS, consistent with previous reports that the AE2 anion exchanger is expressed at the basolateral membrane (Loffing et al. 2000). In contrast, substitution of Cl^– on the apical side had no effect on pH_i in unstimulated cells, but led to a large and rapid rise of 0.60 ± 0.03 (n = 4, P < 0.001) in cells stimulated with 10 μM forskolin. This alkalinization was unaffected by DIDS even at 1 mM. The recovery of pH_i following restoration of apical Cl^– could be mimicked by I^– and formate but not by SO₄^2- or oxalate. RT-PCR performed on RNA extracted from Calu-3 cells revealed transcripts for several members of the SLC26 family. These included pendrin (SLC26A4) which, unlike most other members of the family, is unable to transport divalent anions and is relatively insensitive to DIDS (Mount & Romero, 2004). We therefore propose that pendrin may be the apical anion exchanger in Calu-3 cells and that its activity is markedly stimulated by cAMP, most probably through interaction with CFTR.